1. Genes that Affect the Assembly of the Nervous System in Drosophila Embryos. Genes that affect the assembly of the developing nervous system were screened by RNA interference. Approximately 2,000 double-stranded RNA preparations were injected into embryos and screened. Fourteen genes were found that yield mutant phenotypes that affect the structure of the nervous system. DNA transposition was used to generate mutations in Drosophila genes that affect the structure of the nervous system. Thirty-nine lines of Drosophila were obtained that have recessive lethal mutations in genes that affect the structure of the central nervous system, peripheral nervous system, or both central and peripheral nervous systems. 2. vnd/NK-2 Homeobox Gene. A. Mutation analysis of vnd/NK-2 protein in transgenic lines of Drosophila. Four regions of the vnd/NK-2 homeodomain protein have been conserved during evolution: the homeodomain, tinman motif, an acidic domain, and the NK-2 box. Transgenic lines of Drosophila were generated that ectopically express either wild-type vnd/NK-2 protein or mutant vnd/NK-2 proteins in the ventral, intermediate, or dorsal neuroectoderm and neuroblasts to determine whether deletion of conserved regions of vnd/NK-2 protein affect the ability of the proteins to mediate feedback activation of the vnd/NK-2 gene or repression of ind or msh genes. Replacement of vnd/NK-2 homeodomain Tyr 54 by Met decreased the level of ectopically expressed mutant vnd/NK-2 mRNA, presumably by reducing feedback activation of the vnd/NK-2 gene mediated by vnd/NK-2 protein. The Tyr 54 Met mutant vnd/NK-2 protein had little or no ability to repress ind and msh genes. Deletion of the acidic domain resulted in a relatively small decrease in ectopically expressed mutant vnd/NK-2 mRNA and in a small loss of activity of the mutant protein for repression of ind and msh genes. Deletion of the tinman motif or the NK-2 box had no effect upon feedback activation of the vnd/NK-2 gene or the ability of vnd/NK-2 protein to repress ind or msh genes. The affinity of the Y54M mutant vnd/NK-2 homeodomain for binding to a double-stranded oligodeoxynucleotide with the consensus nucleotide sequence for vnd/NK-2 homeodomain binding was reduced approximately 10-fold compared to the affinity of the wild-type vnd/NK-2 homeodomain. B. Mutation analysis of vnd/NK-2 homeodomain protein using transfection of S2 cells. Thirty-three mutant vnd/NK-2 cDNAs were constructed and were co-transfected into cultured Drosophila S2 cells with DNA containing multiple binding sites for the vnd/NK-2 homeodomain protein linked to a minimal promoter beta-galactosidase reporter gene. Two regions of vnd/NK-2 protein were found to be required for repression of the beta-galactosidase reporter gene and three regions were found that are involved in activation of the reporter gene.